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PubMed2005

Determination of trace proteins with pyronine Y and SDS by resonance light scattering.

A new resonance light scattering (RLS) probe for determining proteins is presented. The weak RLS of pyronine Y-SDS can be enhanced substantially by adding proteins in the presence of H2SO4, resulting in a strong and wide RLS band in the region 310-425 nm. The interaction of pyronine Y-SDS with proteins was studied on the basis of this behavior and a new quantitative method was developed for determining proteins. The enhanced RLS intensity is proportional to the concentration of proteins in the range 0.15-3.6 microg mL(-1) for bovine serum albumin (BSA) and 0.06-4.8 microg mL(-1) for human serum albumin (HSA), with detection limits of 21.0 and 12.0 ng mL(-1), respectively. This method is characterized by high sensitivity, rapidity of reaction, and simplicity. Four synthetic samples were determined satisfactorily and recovery was 99.5-101.5%. Results for human serum and urine samples were in agreement with those obtained by the Bradford method, with relative standard deviations (RSD) of 1.5-3.1%.

Animals↗
PubMed1984

Orthochromatic and metachromatic staining reactions by pyronin Y on Epon semithin sections.

Semithin sections from glutaraldehyde-fixed, Epon-embedded tissues were stained by aqueous solutions of pyronin Y at increasing concentrations (from 10(-6) to 10(-3) mol). Mucopolysaccharide containing structures (e.g. mucin) were found stained in orange, meanwhile the chromatin and remaining tissue components appeared in a bright pink-red color. Cytophotometric measurements showed that a metachromatic shift occurs in the mucin content from goblet cells after pyronin Y staining at 10(-3) mol. Some features of the metachromatic reactions by cationic dyes are briefly discussed.

Animals↗
PubMed1986

Pyronin-phosphotungstic acid: spectral characteristics and staining pattern on Epon semithin sections.

Phosphotungstic acid (PTA) binds to the basic dye pyronin Y to give a non fluorescent complex with absorption characteristics which are different from those of the free dye. The use of this complex on different cells and Epon embedded tissues revealed no staining affinity and only certain hydrated tissue components as starch showed a light reaction with the pyronin-PTA. The role played by the hydratation degree of certain components from epoxy-embedded tissues on the reaction mechanisms observed with some dyes and electron contrasting agents is briefly discussed.

Animals↗
PubMed1990

Kinetic determination of tellurium based on its inhibitory effect on the palladium(II)-catalysed reaction between pyronine G and hypophosphite ion.

A kinetic method for the determination of Te based on its inhibitory effect on the PdII-catalysed reaction between pyronine G and H2PO2- is described. The influence of experimental variables on the rate of the process and the potential interfering effect of a large number of ions has been studied. Under the selected experimental conditions: 6 x 10(-5) M pyronine G; 0.6 M H2PO2-; pH 2.6, adjusted with Britton-Robinson buffer; 0.80 microgram ml-1 of PdII; and a temperature of 22 +/- 0.2 degrees C, Te was determined in the concentration range 0.08-0.85 microgram ml-1. The method was applied to the determination of Te in waters and lead concentrates.

Carbonated Beverages↗
PubMed1988

Iron pyronine and alcian blue for staining acid mucin in plastic-embedded sections.

Iron pyronine Y and Alcian blue demonstrated sulfated and nonsulfated acid mucin, respectively, in plastic-embedded sections. Safran used in combination with these two dyes stained collagen and some reticulum fibers. Sulfated acid mucin was red, while nonsulfated acid mucin stained blue; collagen appeared yellow to greenish yellow. The iron pyronine Y-Alcian blue-safran staining method, when used as in the present article, produces excellent cellular visualization of sulfated and nonsulfated acid mucin.

Alcian Blue↗
PubMed1977

Methyl green-pyronin with hematoxylin and orange G for the identification of inflammatory cells in tissue sections.

Methyl green-pyronin is a notoriously difficult stain to reproduce. Although very useful in detecting cells containing substantial amounts of RNA, it is of limited use in broader problems of cell identification. By careful standardization of the proportions of methyl green to pyronin and combination of these stains with hematoxylin to enhance nuclear contrast and with orange G to improve connective tissue staining, it was possible to produce a consistently reliable staining preparation in which it is possible to identify all the component cells of a mixed inflammatory infiltrate in routine paraffin sections.

Animals↗
PubMed1979

A simple and rapid whole-mount staining method with methyl green-pyronin G applied to a molluscan brain preparation.

Subesophageal ganglia of molluscs have been stained as whole mounts with methyl green-pyronin G to display the relative location of individual neurons. Nuclei appear blue, perikarya red. Expose the ganglion cells by dissection of the connective tissue in snail Ringer. Transfer the ganglion to a fixative of 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.1 at 4 C for 12--24 hours, and wash in distilled water for 1 1/2 hours. Stain with methyl green-pyronin G for 1/2--1 hour and differentiate in 96% ethanol using many rapid changes. Transfer the ganglion to absolute ethanol for 2 1/2 hours and clear in xylene for 3 hours before embedding in Depex in a suitable dish. When the Depex has hardened, the preparation can be stored, and is readily available for subsequent examination. The method may be applicable to other invertebrate tissues, and may be useful in preparing objects for teaching purposes.

Animals↗
PubMed1979

Chromosomal banding patterns produced by methyl green-pyronin staining after trypsin treatment.

A method is described for producing banding patterns with methyl green-pyronin (MGP) stain in chromosomes of fibrosarcoma cells. 1) The stain was made by mixing equal volumes of 2% aqueous pyronin G, 2% aqueous methyl green, distilled water, and 0.1 M acetate buffer (pH 5.7). 2) Treatment with colcemide and hypotonic KCl (0.075 M) was performed as usual. 3) Metaphase chromosomes were prepared using the flame-drying technique and treated with 0.25% trypsin at 37 C for 45 to 90 seconds. Before staining, the slides were rinsed in PBS, in distilled water, and then were dipped in 0.05 M acetate buffer. 4) Chromosomes were stained for more than 20 minutes, rinsed in distilled water, and hot-air dried. Satisfactory results were obtained in uncontracted metaphase chromosomes. MGP stain has the advantage of permitting much longer trypsin treatment and staining time than the trypsin-Giemsa method while providing satisfactory banding patterns.

Animals↗
PubMed1979

[Use of a method of staining sections with methyl green-pyronine for histobacterioscopic purposes].

A method of staining of histological sections with methyl green-pyronine for histobacterioscopic detection of microflora in tissues is proposed. A bright red staining with pyronine of microbial agents and yeast-like fungi of the genus Candida due to a high content of RNA in them permits elective detection of their insignificant amounts in tissues even at low magnifications of the microscope.

Bacteriological Techniques↗
PubMed1971

Effects of coumarin, thiopurines, and pyronin Y on amplification of phleomycin-induced death and deoxyribonucleic acid breakdown in Escherichia coli.

Phleomycin (</=2 mug/ml) induces neither deoxyribonucleic acid (DNA) breakdown nor cell death in stationary-phase Escherichia coli B cells, but the addition of 8 mm caffeine immediately initiates these changes in the same way as increasing the phleomycin concentration 10-fold. This phenomenon is termed "amplification" (6). Pyronin Y, a number of nontoxic thio- and mercaptopurines (of which the most active were 6,7- and 6,9-dimethyl-2-methylthiopurine), and coumarin have been found to be considerably more efficient amplifiers of phleomycin activity than caffeine. Thus 2 mm 6,7- and 6,9-dimethyl-2-methylthiopurine, 0.16 mm pyronin, and 4 mm coumarin killed 10 to 100 times more phleomycin-treated bacteria within 2 hr than 8 mm caffeine. As with caffeine, amplification of cell death by these compounds was accompanied by degradation of DNA to acid-soluble fragments. A number of compounds including 2,6-dichloropurine, 6-hydroxy-2-methylthiopurine, alpha-naphthol, beta-naphthol, naphthionic acid, and alpha-naphthol-4,8-disulphonic acid inhibited the action of phleomycin, if they were present in the cell suspension during phleomycin treatment, but some caused amplification if added subsequent to the phleomycin. Although no mutants resistant to >/=10 mug of phleomycin per ml were observed among 10(11)E. coli B cells screened, such mutants occurred with a frequency of 10(-6) to 10(-7) among cultures resistant to 1 to 2 mug of phleomycin per ml. These double mutants were cross-resistant to phleomycin plus caffeine. The amplifying compounds, though structurally dissimilar, shared the common characteristic of binding selectively to denatured DNA as measured by equilibrium dialysis methods. The implications of these observations in supporting a model of phleomycin amplification proposed previously (6) and their utility in providing a logic for developing a new class of antibiotics are discussed.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

High sensitive fluorophotometric determination of nucleic acids with Pyronine G sensitized by N,N-dimethylformamide.

In Tris-HCl buffer (pH 8.0), the fluorescence of Pyronine G emitted at 552 nm was quenched by nucleic acids when excited at 525 nm. Adding N, N-dimethylformamide (DMF) as a sensitive media can enhance the sensitivity greatly. Based on the fluorescence reactions sensitive fluorimetric methods for nucleic acids at nanogram levels were proposed. Under the optimum conditions, the calibration curves were linear in the range of 0.0032 - 2.5 microg mL(-1) for ct DNA and 0.0024 - 2.5 microg mL(-1) for hs DNA. The limits of determination were 3.2 ng mL(-1) and 2.4 ng mL(-1) respectively. This method has good selectivity and high sensitivity. It has been applied to the determination of DNA in the synthetic samples and real samples with satisfactory results.

Animals↗
PubMed1981

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y.

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.

Acridine Orange↗
PubMed1982

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green.

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.

Animals↗
PubMed1987

Interactions of pyronin Y(G) with nucleic acids.

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.

DNA↗
PubMed1987

Application of pyronin Y(G) in cytochemistry of nucleic acids.

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.

Animals↗
PubMed1984

Spectrophotometric characteristics and assay of pure pyronin Y.

The spectrophotometric characteristics of analytically pure pyronin Y have been investigated. Addition of metal ions (Fe3+, Zn2+, and Mg2+) and of dextrin were shown not to influence the absorption characteristics. The composition of the solvent strongly influenced the value of the extinction coefficient. Aqueous ethanolic solutions with a content of about 50% ethanol gave higher epsilon-values than those found for more concentrated ethanol solutions. The difference can be explained by the existence of a solvent-solute complex in the less concentrated ethanol solutions. A new spectrophotometric assay is proposed using the epsilon-value 11.7 X 10(4) lmol-1 cm-1 found in aqueous ethanol (52%) as standard.

Iron↗
PubMed1979

Chromatin fluorescence by pyronin staining.

Human, chicken and mouse cells from different tissues show a bright red-orange fluorescence of the chromatin after staining with pyronin Y. The possibility that intercalation of the dye into double helical nucleic acids accounts for this fluorescence pattern is briefly discussed.

Animals↗
PubMed2006

Molecular-dynamics simulations of pyronine 6G and rhodamine 6G dimers in aqueous solution.

We have carried out molecular-dynamics (MD) simulations on dimers of the positively charged laser dyes pyronine 6G (P6G) and rhodamine 6G (R6G) in aqueous solution, generating trajectories of 2.5 ns for various computational protocols. We discuss how the choice of atomic partial charges and the length of the trajectories affect the predicted structures of the dimers and compare our results to those of earlier MD-simulations, which were restricted to only 0.7 ns. Our results confirm that monomers of P6G easily undergo relative rotations within the dimer, but we found new conformations of the R6G dimer at longer simulation times. In addition, we analyzed in detail the energy change during the formation of dimers. With suitable corrections, the electrostatic energy from an Ewald treatment agrees with the results from an approach relying on a residue-based cutoff. For P6G, we show that the strong solvent-mediated electrostatic attraction between the monomers is counteracted by an almost equally large solvent-induced entropy contribution to yield a small driving force to dimer formation, in very good agreement with the free-energy change from a thermodynamic-integration procedure. Thus, earlier rationalizations of the dimer formation, based only on energy arguments, yield a qualitatively wrong picture.

Computer Simulation↗